Cardiovascular effects of non-cardiovascular drugs in heart failure
Yurista, Salva
DOI:
10.33612/diss.132706675
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Yurista, S. (2020). Cardiovascular effects of non-cardiovascular drugs in heart failure. University of Groningen. https://doi.org/10.33612/diss.132706675
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Sodium-glucose co-transporter 2 inhibition
with empagliflozin improves cardiac
function in non-diabetic rats with left
ventricular dysfunction after myocardial
infarction
2
Salva R. Yurista
1, Herman H.W. Silljé
1, Silke U. Oberdorf-Maass
1,
Elisabeth-Maria Schouten
1, Mario G. Pavez Giani
1, Jan-Luuk Hillebrands
2,
Harry van Goor
2, Dirk. J. van Veldhuisen
1,
Rudolf A. de Boer
1, B. Daan Westenbrink
11Department of Cardiology, University Medical Center Groningen,
University of Groningen, Groningen, The Netherlands
2Department of Pathology and Medical Biology, Division of Pathology,
University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
2
ABSTRACT
Aims: Sodium glucose co-transporter-2 (SGLT2) inhibition reduces heart failure (HF)
hospitalizations in patients with diabetes, irrespective of glycemic control. We examined the effect of SGLT2 inhibition with empagliflozin (EMPA) on cardiac function in non-diabetic rats with left ventricular (LV) dysfunction after myocardial infarction (MI).
Methods and results: Non-diabetic male Sprague-Dawley rats underwent permanent
coronary artery ligation to induce MI, or sham surgery. Rats received chow containing EMPA that resulted in an average daily intake of 30 mg/kg/day or control chow, starting before surgery (EMPA-early) or 2 weeks after surgery (EMPA-late). Cardiac function was assessed using echocardiography and histological and molecular markers of cardiac remodelling and metabolism were assessed in the left ventricle. Renal function was assessed in metabolic cages.
EMPA increased urine production by two-fold without affecting creatinine clearance and serum electrolytes. EMPA did not influence MI size, but LV ejection fraction (LVEF) was significantly higher in the EMPA-early and EMPA-late treated MI-groups compared to the MI group treated with vehicle (LVEF 54%, 52% and 43% respectively, all P < 0.05). EMPA also attenuated cardiomyocyte hypertrophy, diminished interstitial fibrosis and reduced myocardial oxidative stress. EMPA treatment reduced mitochondrial DNA damage and stimulated mitochondrial biogenesis, which was associated with the normalisation of the myocardial uptake and oxidation of glucose and fatty acids. EMPA increased circulating ketone levels as well as the myocardial expression of the ketone body transporter and two critical ketogenic enzymes, indicating that myocardial utilization of ketone bodies was increased. Together these metabolic changes were associated with an increase in cardiac ATP production.
Conclusion: Empagliflozin favourably affects cardiac function and remodelling in
non-diabetic rats with LV dysfunction after MI, associated with substantial improvements in cardiac metabolism and cardiac ATP production. Importantly, it did so without renal adverse effects. Our data suggest that EMPA might be of benefit in HF patients without diabetes.
Keywords: Diabetes • Heart failure • Metabolism • Mitochondria • Renal function •
2
INTRODUCTION
Type 2 diabetes mellitus (T2DM) and heart failure (HF) commonly coexist.1 Patients with
T2DM have two-fold increased risk of developing HF during their lifetime,2,3 and are also
twice as likely to be hospitalized for HF.4–6
Cardiovascular events and HF hospitalizations can be reduced by adequate glycaemic
control in patients with T2DM,7 but once HF develops the therapeutic options become
limited.8–10 Currently, only metformin is considered safe in HF patients because other oral
anti-diabetic drugs have suspected or established safety concerns in this population.1 This
is not the case for SGLT2 inhibitors (SGLT2i), a new class of anti-diabetic drugs that have been shown to markedly reduce CV events and HF hospitalizations in patients with T2DM. SGLT2i are not only safe and well tolerated in patients with HF but also reduce the incidence
of HF hospitalizations, both in subgroups of patients with and without HF at baseline.11,12
SGLT-2 is a sodium-dependent glucose transporter expressed in the proximal tubule of the kidney and is responsible for 90% of the renal reabsorption of filtered glucose. Accordingly,
SGLT2i induce urinary glucose and sodium excretion and promote diuresis.13 SGLT2i have
relatively few side effects and rarely cause symptomatic hypoglycaemia, even when given
to non-diabetic patients.14 SGLT2i have relatively modest effects on glycaemic control,
suggesting that alternative mechanism may be responsible for the reductions in HF events, which may also apply to non-diabetic patients. The effects of SGLT2i on diuresis and HF hospitalisations without significant side-effects had prompted the hypothesis that SGLT2i could also be of benefit in HF patients, with or without T2DM.
While this hypothesis is currently under investigation in several clinical trials,15,16 the effect
of SGLT2 inhibition on outcomes in non-diabetic HF patients remains unclear at this
point.17,18 We sought to determine the effect of the SGLT2i empagliflozin (EMPA) on cardiac
function and remodelling after MI.
METHODS
A detail description of methods is available in the supplementary Materials.
Experimental protocol
The experimental protocol was approved by the Animal Ethical Committee of University of Groningen (IvD number: 16487-02-001). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
We followed ARRIVE guidelines when reporting this study.19 Non-diabetic male
Sprague-Dawley rats (Envigo, The Netherlands) were randomized to treatment with chow containing EMPA or control chow, starting either 2 days before surgery (early) or 2 weeks after surgery
2
(late). Treatment allocation in the late groups was stratified according to left ventricular ejection fraction (LVEF) 2 weeks post-surgery to ensure that the baseline cardiac function is similar in the EMPA and the vehicle groups. After 10 weeks of treatment, rats were anesthetized, blood was drawn and the hearts were rapidly excised for further analysis. Rats with an infarct size of less than 15% were excluded from analysis as these small infarcts
are haemodynamically fully compensated.20
MI surgery
Rats were randomized to permanent ligation of the left anterior descending coronary artery
or sham surgery under isoflurane (2.5%) inhalation anaesthesia, as previously described.21
Investigational drug
Empagliflozin (BI 10773) was kindly supplied by Boehringer Ingelheim, Germany. EMPA (BI 10773) was mixed with standard rat chow (R/M-H V1534-70, Ssniff, Germany) in a final concentration of 200 mg/kg intended to reach an average dose of 30 mg/kg/day.
Echocardiography
Two weeks after surgery and 1 week before termination, the M-mode and two-dimensional echocardiography was performed using a Vivid 7 echo machine (GE Healthcare, Milwaukee, WI, USA) equipped with a 10-MHz phase array linear transducer for serially assessment of
cardiac structure and function as previously described.21,22
Invasive hemodynamic measurements
Prior to sacrifice, invasive haemodynamics were analysed by aortic and left ventricular
(LV) catheterization, as previously described.21 The data were acquired using a PowerLab
data acquisition system (ADInstruments, Colorado Springs, CO, USA) and analyzed with a LabChart 8 software.
Infarct size, cardiomyocyte size and interstitial fibrosis measurement
For immunohistochemical analysis, the mid-papillary slice of the left ventricle was fixed in 4% formaldehyde and paraffin-embedded. Masson trichrome staining was performed to evaluate the infarct size and the extent of interstitial fibrosis. Furthermore, to determine cardiomyocyte size, sections were stained with FITC Labelled wheat germ agglutinin (WGA)
as previously described.21,22
Metabolic cage
Two weeks before termination, rats were placed in metabolic cages to monitor 24-hour
2
Blood and urine measurementsBlood samples were obtained via tail vein at regular intervals under isoflurane anaesthesia to monitor blood glucose and haematocrit levels. At sacrifice, 8 ml of blood was drawn from the abdominal aorta (either anti-coagulated with EDTA or sodium heparin), and urine was collected directly from bladder.
Mitochondrial DNA (mtDNA)-to-nuclear DNA (nDNA) ratio and mtDNA damage
Total DNA including mtDNA was extracted from the non-infarcted left ventricle using Nucleospin® Tissue XS (Macherey-Nagel GmBH&Co. KG, Düren, Germany). mtDNA-to-nDNA ratio was determined by quantitative real-time polymerase chain reaction
(qRT-PCR), as described previously.24 Expression of mitochondrial genes were corrected
for nuclear gene expressions values, and the calculated values were expressed relative to the control group per experiment. To determine DNA damage (lesions/10kb), the D-loop mitochondrial genomic region was amplified by a semi-long-run qRT-PCR, as described
before.24 Primer sequences are listed in the supplementary Table S1.
Quantitative real-time polymerase chain reaction
RNA was extracted from the non-infarcted left ventricle using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) and QuantiTect RT kit (Qiagen) was then used to make cDNA,
following manufacturer’s instructions as previously described.25 36B4 reference gene
were used to correct all measured mRNA expression. Primer sequences can be found in supplementary Table S1.
Advanced oxidation protein products measurements
The advanced oxidation protein product (AOPP) assay was performed using the AOPP Assay Kit from Abcam (#ab242295, Cambridge, U.K.) according to the manufacturer’s instructions. Results were then normalized by protein concentrations of each test.
ATP measurements
The ATP assay was performed using the ATP Assay Kit (colorimetric/fluorometric) from Abcam (#ab83355, Cambridge, U.K.) according to the manufacturer’s instructions. Results were then normalized by protein concentrations of each test.
Pyruvate dehydrogenase activity
According to manufacturer’s instruction, we measured the activity of pyruvate dehydrogenase (PDH) using the assay kit from Sigma MAK183 and a spectrophotometric multiwell plate reader (Synergy H1, BioTek).
2
Insulin and glucagon measurements
Circulating hormones were measured by a rat ultrasensitive insulin ELISA kit (80-INSRT-E01, ALPCO) and Glucagon ELISA (48-GLUHU-E01, ALPCO) according to the manufacturer’s instructions.
Statistical analysis
Data are presented as means ± standard errors of the mean (SEM). To compare normally
distributed parameters,one-way analysis of variance (ANOVA) followed by Tukey’s Post
Hoc test was used. When data were not normally distributed, a non-parametric Kruskal-Wallis test followed by a Mann-Whitney U test with correction for multiple comparisons was used. To compare EMPA and Vehicle treatment independent of treatment allocation, an independent T test or a Mann-Whitney U test was used, where appropriate. Wilcoxon signed rank test was used to evaluate LVEF post-MI versus before termination (Figure 2E). Differences were considered significant at p<0.05. IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY, USA) was used to perform all statistical analysis.
RESULTS
A total of 140 rats were randomized to MI or sham surgery, 47 rats died during the surgical procedure. Overall mortality in the MI groups was 41%. All rats died from ventricular fibrillation within 60 minutes of left anterior descending coronary artery ligation. No mortality was observed during the subsequent stages of the study and no mortality was observed in sham-operated rats throughout the study. There were no statistically significant differences in mortality between the MI-vehicle vs. the MI-EMPA-E or the MI-EMPA-L groups (43% vs. 40% vs. 40%, respectively). In total, 20 rats with infarct sizes of < 15% were excluded from analysis (8 rats in MI-vehicle group, 5 rats in MI-EMPA-E group and 7 rats in EMPA-L group), leaving a total of 73 rats for the current analysis. The final group sizes were 8 for the sham-vehicle group, 19 for sham-EMPA group, 22 for vehicle group, 13 for MI-EMPA-early group and 11 for MI-EMPA-late group.
Efficacy and safety of empagliflozin in non-diabetic rats with LV dysfunction after MI Efficacy of empagliflozin
Daily food intake was comparable between EMPA and vehicle treated groups and the average daily dose of EMPA was 30 mg/kg of body weight/day (supplementary Figure S1A). As expected, EMPA increased urinary glucose (Figure 1A) and sodium excretion (Figure 1B), resulting in a 2-fold increase in urine production (Figure 1C). The increase in urinary glucose excretion was associated with a substantial reduction in body weight in the EMPA treated sham and MI groups (supplementary Figure S1C).
2
SafetyThe increase in diuresis after EMPA treatment was compensated by a proportionate increase in fluid intake (Supplementary Figure S1B) and did not affect haematocrit (supplementary
Figure S2A) or renal function (Figure 1D), indicating that EMPA did not cause excessive
plasma contraction. In addition, EMPA did not cause significant changes in plasma glucose, sodium or potassium levels (supplementary Figure S2B-D).
24h -u ri n ar y g lu co s e e xcr et io n ( m m o l) V e h EM P A V e h EM P A -E EM P A -L 0 5 1 0 1 5 * * # S h a m M I 2 4 h-ur ina ry s od ium e xcr et io n ( m m o l) V e h EM P A V e h EM P A -E EM P A -L 0 1 2 3 4 # * * S h a m M I 2 4 h-u ri ne pr od uc ti o n (m l) V e h EM P A V e h EM P A -E EM P A -L 0 1 0 2 0 3 0 4 0 5 0 # * * S h a m M I C reat in in e cl ear an c e (m l/m in /k g ) V e h EM P A V e h EM P A -E EM P A -L 0 5 1 0 1 5 S h a m M I Figure 1. A C B D
FIGURE 1. Efficacy and safety of empagliflozin (EMPA) in non-diabetic rats with left ventricular dysfunction after myocardial infarction (MI). (A) 24 h urinary glucose excretion. (B) 24 h urinary sodium excretion. (C) 24 h urine production. (D) Creatinine clearance of all groups. EMPA-E, EMPA-early; EMPA-L, EMPA-late; Veh, vehicle. Data are presented as means ± standard errors of the mean. *p<0.05 vs. MI-Veh; #p<0.05 vs. Sham-Veh.
Effect of empagliflozin on blood pressure and cardiac function
The average LV infarct size was 33% and did not differ among the MI, MI-EMPA-early and MI-EMPA-late groups (Figure 2A and 2B). Systolic and diastolic blood pressure was also not different between the groups (supplementary Table S2). As expected, MI resulted in significant LV dilatation (Figure 2C) and a reduction in LVEF (Figure 2D). LVEF was significantly higher in both MI-EMPA treated groups compared to the MI-vehicle group (Figure 2D) A pooled analysis of longitudinal changes in LVEF revealed that EMPA prevented the progressive deterioration of cardiac function that occurs after MI (Figure 2E). Other relevant echocardiographic parameters are depicted in supplementary Table S3. There was no significant between-group difference in LV filling pressures (supplementary Table S2).
2
In fa rc t s iz e ( %) Ve h EM P A Ve h E M P A -E E M P A -L 0 10 20 30 40 S h am MI P o st-M I B ef o re t er m in at io n 0 30 40 50 60 70 Eje cti on fr ac tio n ( %) M I-V e h MI -E MP A ‡ Masso ns trich rom eB
A
E
Fi gu re 2.C
Sha m -V eh Sha m -E MP A MI -V eh MI -EM PA -E M I-EM PA -LD
F
LV ID d ( mm ) Ve h EM P A Ve h E M P A -E E M P A -L 0 8 10 # S h am MI Eje cti on F ra cti on (% ) Ve h EM P A Ve h E M P A -E E M P A -L 0 40 50 60 70 80 * * # S h am MI Ve nt ric ula r w eig ht / Tib ia le ng th (m g/m m) Ve h EM P A Ve h E M P A -E E M P A -L 0 25 30 35 S h am MI * * # FIGURE 2 . E ffect o f empagliflo zin (EMP A) o n c ar dia c fu nctio n in no n-diabetic rats with left v
entricula r d ysfu nctio n a fter m yoc ar dial in fa rctio n (MI). (A) R ep resentati ve left ventricula r sectio ns stained with M asso ns trich ro me . (B) Qua ntific atio n o f in fa rct siz e fr om M asso ns trich ro me stained sectio n. (C) L eft v entricula r int ernal dimensio ns in diast ole (L VIDd). (D) L eft v entricula r ejectio n fr actio n. (E) L ongitu dinal cha nge o f left v entricula r ejectio n fr actio n post -MI a nd bef or e t erminatio n. (F) R atio o f v entricula r w eight t o tibia length. EMP A-E, EMP A-ea rly; EMP A-L, EMP A-lat e. V eh, v ehicle . Data a re p resent ed as mea ns ± sta nda rd err ors o f the mea n. * p<0.05 vs . MI -V eh; # p<0.05 vs . S ha m-Veh; ‡ p<0.05 vs . MI -V eh post -MI .
2
Effect of empagliflozin on cardiac histology and molecular markers for remodellingand fibrosis
A large MI invariably causes pathological remodelling of the non-infarcted myocardium
which contributes to further deterioration of cardiac performance.26–28
Marked cardiac hypertrophy was observed after MI as reflected by a 10% increase in ventricular mass (Figure 2F) and an 81% increase in cardiomyocyte cross sectional area (Figure 3A and 3B). Both early and late treatment with EMPA attenuated the increase in left ventricular mass (Figure 2F) and diminished cardiomyocyte cross-sectional area compared to the vehicle-treated MI group (Figure 3A and B). Myocardial fibrosis was increased three-fold in the non-infarcted left ventricle, and this was also was markedly attenuated by EMPA treatment (Figure 3B and 3C). The reductions in fibrosis after EMPA treatment were accompanied by similar reductions in the expression of the fibrosis markers collagen 1 and procollagen (Figure 3E).
Increased myocardial expression of atrial natriuretic peptide (ANP) and an increase in the relative expression of foetal (β-MHC) over adult (α-MHC) myocin heavy chain isoform ie. β-MHC/α-MHC ratio are generally considered as robust markers for the activation of the
cardiac fetal gene program.29 Myocardial ANP expression was increased by six-fold in the
vehicle-treated MI group compared to the sham groups. Both early and late treatment with EMPA reduced cardiac ANP expression by 50%. Similarly, the increase in β-MHC/α-MHC ratio observed in the MI group was normalized to sham levels after EMPA treatment (Figure 3D).
Effects of empagliflozin on oxidative stress
To investigate the effect of EMPA on myocardial oxidative stress, we determined the advanced oxidation protein products (AOPP) level and the expression level of the superoxide generating enzyme NOX2. AOPP was higher in the heart of rats in MI-vehicle group than in the sham-vehicle group, and was suppressed by EMPA in both early and late treatment (Figure 4A). Similarly, expression of NOX2, was up-regulated in MI-vehicle group, but was attenuated in both the MI-EMPA-early and MI-EMPA-late group (Figure 4A).
2
C o ll a g e n I P ro co ll a g e n 0 .0 0 .5 1 .0 1 .5 2 .0 mR NA le ve l ( fo ld c ha ng e) # * * # * * ANP β/ α -M H C r a ti o 0 2 4 6 8 mR NA le ve l ( fo ld c ha ng e) # * * # * * % F ibro sis Ve h EM P A Ve h E M P A -E E M P A -L 0 5 10 15 20 25 * * # S h am MI Ca rd io my oc yte cro ss -s ec tio na l a re a ( µ m 2 ) Ve h EM P A Ve h E M P A -E E M P A -L 0 200 400 600 800 1000 * * # S h am MI Whe at g erm in(WG lutin aggA) e rom trich ns Masso
C
B
Fi gu re 3.A
D
Sha m -V eh Sha m -E MP A MI -V eh MI -EM PA -E M I-EM PA -LE
Sh a m -Ve h Sh a m -EM P A M I-V e h M I-E M P A -E M I-EM PA -L FIGURE 3 . E ffect o f empagliflo zin (EMP A) o n pathologic al c ar dia c r emodelling a nd fib rosis in no n-diabetic rats with left v
entricula r d ysfu nctio n a fter m yoc ar dial in fa rctio n (MI). (A) Qua ntific atio n o f c ar dio m yocyt e c ross-sectio nal a rea fr
om wheat germ agglutinin (W
GA) stained sectio
n. (B) R ep resentati ve left v entricula r sectio ns stained with W GA a nd M asso ns trich ro me staining to assess c ar dio m yocyt e h ypertr oph y a nd fib rosis . (C) Qua ntific atio n of fib rosis in the no n-in fa rct ed left ventricle fr om M asso ns trich ro me stained sectio n. (D ,E) M easu rement o f m RNA lev els t o assess molecula r ma rk ers f or r emodelling a nd fib rosis , r especti vel y, no rmaliz ed t o 36B4. EMP A-E, EMP A-ea rly; EMP A-L, EMP A-lat e; Veh, v ehicle . Data a re p resent ed as mea ns ± sta nda rd err ors o f the mea n. * p<0.05 vs . MI -V eh; # p<0.05 vs . S ha m-Veh.
2
Effects of empagliflozin on mitochondrial biogenesis and ATP productionDetailed cardiac and renal analysis of diabetic animal models has revealed tha the SGLT2i
affect mitochondrial morphology and mitochondrial density.30,31 Mitochondrial content
and respiratory capacity are also significantly reduced in HF, which is tied to diminished
expression of the transcription factor PGC-1α.32 To determine whether the favourable effects
of EMPA on cardiac remodelling were associated with similar recovery of mitochondrial biogenesis, mitochondrial content was quantified by normalizing mtDNA to nDNA. We also performed a semi-long-run PCR to detect mtDNA damage. As expected, mtDNA damage was also increased after MI (Figure 4B). In addition, mtDNA/nDNA was markedly reduced after MI (Figure 4C), accompanied by similar reductions in PGC-1α expression (Figure 4C). EMPA normalized PGC-1α expression and partial recovered of mtDNA/nDNA (Figure 4A) and mtDNA damage (Figure 4B).
To verify whether the effects of EMPA on mtDNA damage and the increase in mtDNA/nDNA were associated with increased ATP production, we determined the cardiac ATP levels in our study. ATP levels were significantly reduced in MI-vehicle group and interestingly, ATP levels were significantly restored in MI-EMPA-early group and there was a trend towards increased ATP levels in the MI-EMPA-late group (p=0.08) (Figure 4D).
Effects of empagliflozin on myocardial glucose and fatty acid metabolism
HF is accompanied by profound changes in the myocardial utilization of carbon based
fuels.33 During HF development, cardiac substrate preference first shifts from fatty acids
to glucose as the primary fuel source, while the later stages of HF are also associated
with disruption of myocardial glucose uptake and utilization.33 Molecular signatures
for impaired substrate utilization are, among others, down-regulation of the fatty acid transporter carnitine palmitoyltransferase I-α (CPT1-α), downregulation of the glucose transporter type 4 (GLUT4) and inhibition of the pyruvate dehydrogenase complex through
up-regulation of pyruvate dehydrogenase kinase 4 (PDK4).33 Pyruvate dehydrogenase is
required for carbohydrate intermediates to enter the Krebs cycle and PDK4 inhibits this
enzyme complex.33
As expected, CPT1-α, GLUT4, PGC1-α levels and PDH activity were reduced and PDK4 expression was increased in cardiac tissue of MI-vehicle group compared to sham-vehicle group. Interestingly, EMPA treatment restored the expression of all these markers to sham levels (Figure 5A-5B, 5D), suggesting that EMPA restores myocardial substrate metabolism.
2
C ar d ia c A T P (n m o l/m g p ro te in ) V e h EM P A V e h EM P A -E EM P A -L 0 2 4 6 8 1 0 S h a m M I * # V e h EM P A V e h EM P A -E EM P A -L 0 5 1 0 1 5 2 0 2 5 M it oc hondr ia l D N A l e s ion/ 1 0 k b S h a m M I * * # R e la tiv e m tD N A /n D N A P G C 1 -α 0 .0 0 .5 1 .0 1 .5 M it oc ho nd ri a l B io ge ne s is # * * # * * C a rd ia c A O P P N O X 2 0 1 2 3 O xi d at ive st ress (f ol d c ha nge ) # * * # * * B A Figure 4. D C S h a m - V e h S h a m -E M P A M I- V e h M I-E M P A -E M I-E M P A -L S h a m - V e h S h a m -E M P A M I- V e h M I-E M P A -E M I-E M P A -LFIGURE 4. Effect of empagliflozin (EMPA) on oxidative stress, mitochondrial biogenesis and cardiac ATP levels. (A) Cardiac advanced oxidation protein products and measurement of mRNA levels to assess oxidative stress. (B) Semi-long-run polymerase chain reaction was performed using primers specific for mitochondrial DNA fragments in the mitochondrial D-loop region to determine mitochondrial DNA damage. (C) Mitochondrial DNA (CYTB)-to-nuclear DNA (TRPM-2) ratio and mRNA levels to assess mitochondrial biogenesis. (D) Cardiac ATP levels. E, EMPA-early; EMPA-L, EMPA-late; MI, myocardial infarction; Veh, vehicle. Data are presented as means ± standard errors of the mean. *p<0.05 vs. MI-Veh; #p<0.05 vs. Sham-Veh.
Effects of empaglilfozin on ketone metabolism
The disruption of glucose and fatty acid oxidation causes the failing heart to increasingly rely
on ketone bodies as a fuel source.34,35 EMPA increases circulating ketone levels and indirect
evidence suggests that EMPA promotes ketone utilization in patients with diabetes.36
The beneficial effects of EMPA on cardiac performance could therefore be explained by
2
C P T 1-α m R N A l e v e l V e h EM P A V e h EM P A -E EM P A -L 0 .0 0 .5 1 .0 1 .5 2 .0 S h a m M I * * # Ins ul in / G luc a g on ra ti o V e h EM P A V e h EM P A -E EM P A -L 0 2 4 6 S h a m M I * * # V e h EM P A V e h EM P A -E EM P A -L 0 1 5 0 2 0 0 2 5 0 P D H a ct iv it y ( n m o l/m in /m g ) S h a m M I * * # G L U T 4 P D K 4 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 m R N A l e v el ( fo ld c h a n g e ) # * * # * * B A Figure 5. D C S h a m - V e h S h a m -E M P A M I- V e h M I-E M P A -E M I-E M P A -LFIGURE 5. Effect of empagliflozin (EMPA) on glucose metabolism, insulin/glucagon ratio and fatty acid metabolism. (A) Measurement of mRNA levels to assess cardiac glucose metabolism. (B) Pyruvate dehydrogenase (PDH) activity. (C) Insulin/glucagon ratio. (D) Measurement of mRNA levels to assess fatty acid metabolism. CPT1-𝛼, carnitine palmitoyltransferase 1-𝛼; EMPA, empagliflozin; EMPA-E, EMPA-early; EMPA-L, EMPA-late; MI, myocardial infarction; Veh, vehicle. Data are presented as means ± standard errors of the mean. *p<0.05 vs. MI-Veh; #p<0.05 vs. Sham-Veh.
Hepatic ketogenesis is induced by reductions in the systemic insulin/glucagon ratio and EMPA decreased insulin-to-glucagon ratio (Figure 5C). Consistent with these observations, EMPA treatment increased both circulating ketone levels and urinary ketone excretion, in sham and MI groups (Figure 6A and 6B). To determine whether the increases in circulating ketone levels was also associated with changes in the myocardial capacity to utilize ketone bodies we performed a detailed analysis of three critical proteins involved in myocardial ketolysis, namely the ketone body transporter monocarboxylate transporter 1 (MCT1), ketogenic enzyme β- hydroxy butyrate dehydrogenase (BDH1) and Succinyl-CoA:3-ketoacid CoA transferase (SCOT). The expression of the MCT1 and BDH1 gene and the protein expression of SCOT were all significantly increased in MI-vehicle group. Furthermore, the expression was further increased in both MI-EMPA-early and MI-EMPA-late groups (Figure
6C and 6D). These findings suggest that ketone bioavailability and cardiac ketone utilization
2
S C O T p ro tei n l evel s (f ol d c ha nge ) V e h EM P A V e h EM P A -E EM P A -L 0 1 2 3 4 S h a m M I * * # M C T 1 B D H 1 0 1 2 3 6 m R N A l e v el ( fo ld c h a n g e ) # * * # * * 2 4 h-u ri na ry k e tone b od y excr et io n ( µ m o l) V e h EM P A V e h EM P A -E EM P A -L 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 # * * S h a m M I P la sm a to ta l k e to n e b o d y (m m o l/l) V e h EM P A V e h EM P A -E EM P A -L 0 .0 0 .1 0 .2 0 .3 0 .4 S h a m M I * * # B A C Figure 6. D S h a m - V e h S h a m -E M P A M I- V e h M I-E M P A -E M I-E M P A -LFIGURE 6. Effect of empagliflozin (EMPA) on ketone metabolism. (A) Plasma total ketone body levels. (B) 24 h urinary ketone body excretion. (C) Measurement of mRNA levels to assess ketone metabolism. (D) Protein levels of succinyl-CoA:3-ketoacid CoA transferase (SCOT). BDH1, ketogenic enzyme 𝛽-hydroxy butyrate dehydrogenase; EMPA-E, EMPA-early; EMPA-L, EMPA-late; MCT1, monocarboxylate transporter 1; MI, myocardial infarction; Veh, vehicle. Data are presented as means ± standard errors of the mean. *p<0.05 vs. MI-Veh; #p<0.05 vs. Sham-Veh.
DISCUSSION
In the present study in which we treated non-diabetic rats with LV dysfunction after a large MI with the SGLT2i EMPA for 10 weeks, diuresis was markedly increased without affecting kidney function, serum glucose and electrolyte levels. EMPA did not influence MI size, but it did improve cardiac function and attenuated pathological cardiomyocyte hypertrophy and cardiac fibrosis. The beneficial effects of EMPA on cardiac function were associated with favourable effects on cardiac metabolism, including reduction of mitochondrial DNA damage and oxidative stress, activation of mitochondrial biogenesis, and the restoration of cardiac glucose and fatty acid oxidation. Furthermore, EMPA promoted the bioavailability of ketones as well as the cardiac capacity of utilize ketone bodies as a fuel source. These findings provide robust evidence that EMPA improves cardiac performance in non-diabetic failing hearts, and provides further mechanistic insights that provide a rationale for the clinical trials that are underway.
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SGLT2i have emerged as oral anti-diabetic drugs that reduce cardiovascular events and
HF hospitalisations in patients with diabetes.11,12,39 Pre-specified secondary analysis of the
Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients (EMPA-REG OUTCOME) revealed that EMPA reduced new-onset HF and that the reduction in HF hospitalizations was consistent among several subgroups, including patients with
HF at baseline.11 In the Canagliflozin Cardiovascular Assessment Study (CANVAS), SGLT2i
canagliflozin also reduced HF hospitalizations compared to placebo and the reductions in
HF hospitalizations were more pronounced in patients with a history of HF at baseline.12
It has to be noted that the history of HF was not robustly adjudicated and that the results of these secondary analyses should be considered hypothesis generating. The validity of this hypothesis is currently being tested in dedicated cardiovascular outcome trials in HF patients with reduced and preserved ejection fraction (NCT03057977, NCT03057951, NCT03036124, NCT03619213), which are expected to be completed in 2019-2022.
Interestingly, these trials have been launched in the absence of robust evidence that SGLT2i affect cardiac performance in the absence of diabetes. Indeed, most mechanistic studies
have been performed in the context of diabetes. Byrne et al.40 reported that EMPA treatment
prevented the progressive deterioration of cardiac dysfunction in a model of transverse aortic constriction surgery in vivo and ex vivo. Another study reported that EMPA reduced
cardiac hypertrophy and fibrosis in rat model of metabolic syndrome with prediabetes.41
Moreover, EMPA was also shown to improve diastolic function and ameliorate cardiac
hypertrophy and fibrosis in female db/db mice.42 Evidence for clinical translation of
these findings was recently provided by the EMPA-HEART Cardiolink-6 study, which demonstrated that 6 months of treatment with EMPA resulted reduced cardiac mass and increased LVEF (Verma S., unpublished data)
In non-diabetic context, a study of Lee et al.43 demonstrated that the SGLT2i dapagliflozin
and phlorizin attenuated oxidative stress after myocardial infarction in non-diabetic male Wistar rats. They also observed that SGLT2i did not reduce the infarct sizes, however it
did attenuate the cardiac fibrosis.43 Our data extends this observation as we demonstrate a
reduction in fibrosis and oxidative damage to mitochondrial DNA. In addition, we have now added evidence that EMPA also attenuates total myocardial oxidative stress as evidenced by reductions in AOPP and NOX2 expression (Figure 4A).
The mechanisms responsible for the beneficial effects of SGLT2i on HF outcomes are a
matter of intense speculation.15,18 The most obvious beneficial mechanisms could be derived
from the fact that SGLT2i are potent proximal tubule diuretics. The diuretic effects of EMPA were obvious from our study and, in contrast to other diuretics, EMPA did not affect renal function and plasma levels of glucose and electrolytes remained normal. These findings are
consistent with the reno-protective effects observed in patient with diabetes.44,45 Of note,
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is no data to support a prognostic benefit of using diuretics in this population.8 Another
interesting hypothesis is that the natriuresis induced by EMPA attenuates sodium overload in cardiomyocytes through inhibition of the sarcolemmal sodium-hydrogen exchanger. Elevated intracellular sodium levels are thought to compromise mitochondrial calcium
handling and thereby contributes to mitochondrial dysfunction in HF.46 The metabolic
effects of EMPA observed in our study could thus partially reflect reductions in intracellular sodium. Nevertheless, it is hard to comprehend that the profound effects of EMPA on cardiac remodelling and cardiac metabolism that we observed are solely explained by their diuretic effects, as we did not observe a difference in blood pressure or LV filling pressures (Supplementary Table S2).
Another possible explanation for the beneficial effects of SGLT2i could lie in their effects on systemic and cardiac metabolism, which have consistently been observed in models of diabetes and heart disease. First, EMPA improves glycaemic control in diabetic patients and the caloric loss associated with increased urinary glucose excretion results in weight
loss.47,48 Weight loss is thought to improve myocardial insulin sensitivity and myocardial
insulin resistance is common in severe HF, irrespective of the presence of T2DM.33
Second, several studies have suggested that SGLT2i can restore cardiac mitochondrial dysfunction. Indeed, the SGLT2i dapagliflozin restored cardiac PGC1-α in prediabetic
rats.49 PGC1-α is a critical mediator of mitochondrial biogenesis and the reductions in
PGC1-α are thought to contribute to mitochondrial dysfunction observed in HF. Mizuno
et al.30 recently demonstrated that EMPA normalizes the size and number of mitochondria in diabetic hearts after a MI. This is consistent with our observation that EMPA restored the mtDNA damage, mtDNA/nDNA ratio and restored PGC1-α expression, indicating that the effects of SGLT2i on mitochondrial biogenesis can be translated to the non-diabetic failing heart. Third, the changes in PDH activity and the expression levels of CPT1, GLUT4
and PDK4 observed by us and by others,49 suggest that the myocardial capacity to oxidize
glucose and fatty acids is improved by EMPA. Verma et al.50 recently validated this concept
by comparing substrate utilisation in a working heart model using hearts from EMPA or vehicle-treated diabetic rats. In accordance with our observations, these authors also showed that EMPA treatment restored myocardial glucose and fatty acid oxidation to non-diabetic levels. Fourth, during disease progression non-diabetic, and failing hearts increasingly
rely on ketone bodies as a fuels source,37,38,51 and SGLT2i increase circulating ketone levels.36
The myocardial capacity to metabolize ketone bodies is increased in HF, reflected by an
increased expression of ketogenic enzymes such as BDH1.37,38 Ketones are considered to
be more energy efficient under conditions of metabolic stress as they require less oxygen
per molecule of ATP generated.37,38 In addition, ketone oxidation does not inhibit the
oxidation of other substrates through the Randel cycle,50 and ketones appear to improve
myocardial blood flow.52 A single dose of EMPA was recently shown to increase myocardial
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findings that EMPA increases the bioavailability and the myocardial utilization of ketone bodies which also increases ATP production in non-diabetic rats with LV dysfunction after MI are consistent with these observations. Together these data strongly suggest that the restoration of cardiac ATP levels by EMPA is at least partially caused by increased ketones oxidation. Of note, the result suggests that SGLT2-mediated refuelling of the heart contributes to the beneficial effects of EMPA on cardiovascular outcomes. Our study thus clearly demonstrates that the salutary effects of EMPA on cardiac metabolism that have been described in diabetic models also occur in a non-diabetic context.
STUDY LIMITATIONS
First, our study provides a detailed insight into the myocardial effects of SGLT2 inhibition but was not designed to establish cause and effect of the proposed mechanisms responsible for the cardio-metabolic effects. Second, while we describe multiple molecular effect of EMPA in the myocardium, we did not establish the mechanisms underlying these molecular changes. More focused mechanistic studies with for instance BDH1 or SCOT knockout mice
would be required for this purpose.54 Third, we also do not provide direct proof that ketone
body oxidation is increased in EMPA-treated hearts nor did we quantify the contribution of ketone body oxidation to the increase in myocardial ATP levels. This would require detailed metabolomics analysis, which is beyond the scope of the current manuscript. In addition, the effects of EMPA on ketone body utilization appear to be dependent on the severity of
cardiac dysfunction as myocardial ketone utilization is not affected in mild HF.50,55 Ketone
bodies may also have vasodilatory effects on the myocardium, which could explain part of
the protective effects beyond myocardial ketone utilization.52 Finally, while the post-MI LV
dysfunction models allows robust and predictable changes in cardiac remodelling and HF development, the effects of EMPA may be different in a clinical context. Nevertheless, our study provides unique insights into the cardiac effects of EMPA in the non-diabetic failing heart and supports the exploration of EMPA as a bona fide HF drug in patients with or without diabetes.
CONCLUSION
Empagliflozin favourably affects cardiac function and remodelling in non-diabetic rats with LV dysfunction after MI, associated with substantial improvements in cardiac ATP production. Importantly, it did so without renal adverse effects. Our data suggest that EMPA might be of benefit in HF patients without diabetes.
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ACKNOWLEDGEMENTS
We acknowledge Boehringer Ingelheim for supplying empagliflozin and control chow, and thank Dr. Eric Mayoux for support and expert advice throughout the project. We thank Janny Takens and Martin Dokter for expert technical assistance and advice.
FUNDING
Dr. Yurista is supported by a grant from the Indonesia Endowment Fund for Education (LPDP No. 20150722083422). Dr. de Boer is supported by the Netherlands Heart Foundation (CVON DOSIS, grant 2014-40, CVON SHE-PREDICTS-HF, grant 2017-21, and CVON RED-CVD, grant 2017-11); and the Innovational Research Incentives Scheme program of the Netherlands Organization for Scientific Research (NWO VIDI, grant 917.13.350). Dr. Westenbrink is supported by The Netherlands Organisation for Scientific Research (NWO VENI, grant 016.176.147).
CONFLICT OF INTEREST
The UMCG, which employs all authors, has received research grants and/or fees from AstraZeneca, Abbott, Bristol-Myers Squibb, Novartis, Roche, Trevena, and ThermoFisher GmbH. Dr. de Boer is a minority shareholder of scPharmaceuticals, Inc. Dr. de Boer received personal fees from MandalMed Inc, Novartis, and Servier. The other authors do not report conflict of interest.
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SUPPLEMENTARY MATERIALS
Supplementary Methods Animals
The investigation was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The experimental protocol was approved by the Animal Ethical Committee of University of Groningen (IvD number: 16487-02-001). The study was performed in non-diabetic male Sprague Dawley rats weighing 250-280 g (Envigo, The Netherlands). Animals were fed ad libitum and housed conventionally in groups of two to four rats with 12:12 h light-dark cycles (N = 8-22/group, unless indicated). We followed ARRIVE guidelines when reporting this study.
Myocardial infarction surgery
Rats were randomized to HF or sham surgery under isoflurane (2.5%) inhalation anaesthesia. After left-sided thoracotomy, HF was induced by permanent ligating of the proximal portion of the left coronary artery. Sham operated rats underwent the same procedure but without coronary ligation.
Experimental protocol
We aimed to study the effect of empagliflozin (EMPA) on both early remodelling after an acute MI, as well as the effect of EMPA on the already remodelled heart and chose two treatment regimens. In the early treatment group, rats were randomized to EMPA or vehicle starting 2 days before surgery and continued until 10 weeks after MI. In the late group, rats were stratified according to left ventricular ejection fraction and then randomized to EMPA or vehicle starting 2 weeks after MI or sham surgery. After 10 weeks of treatment, rats were anesthetized, blood was drawn (either anti-coagulated with EDTA or sodium heparin) and the hearts were rapidly excised. Myocardial tissue was sectioned transversally and processed for immunohistochemistry or snap-frozen for molecular analysis. Rats with an infarct size of less than 15% were excluded from analysis as these small infarcts are haemodynamically fully compensated.
Echocardiography
Two weeks after surgery and 1 week before termination, the M-mode and 2D echocardiography was performed using a Vivid 7 echo machine (GE Healthcare) equipped with a 10-MHz phase array linear transducer for serially assessment of cardiac structure and function. The measurements included systolic and diastolic LV internal dimensions (LVIDs and LVIDD). LV fractional shortening was calculated as FS = (LVIDd – LVIDs) / LVIDd x 100 %. LV ejection fraction (LVEF%) was calculated by using the Teichholz method of estimated LV volumes.
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Invasive hemodyamic measurements
Prior to sacrifice, invasive hemodynamics were analysed by aortic and LV catheterization. The right carotid artery was isolated, punctured, and a 1.9 F rat pressure‐volume catheter (Scisense, London, Ontario, Canada) was inserted into the right carotid artery. The tip of the catheter was advanced through the aorta into the LV cavity. Heart rate (HR), left ventricular end-systolic (LVESP) and end-diastolic (LVEDP) pressures, and maximal rates of increase and decrease in developed LV pessures (dP/dtmax and dP/dtmin) were determined. The data were acquired using a PowerLab data acquisition system (ADInstruments, Colorado Springs, CO) and analyzed with a LabChart 8 software.
Infarct size, cardiomyocyte size and interstitial fibrosis measurement
Rats were euthanized under isoflurane anaesthesia. Heart were rapidly excised and weighed. The mid-papillary slice of the LV was fixed in 4% formaldehyde and paraffin-embedded. Infarct size was calculated as percentage of the scar length to the total LV circumference on Masson trichrome-stained section. Furthermore, Masson trichrome staining was also used to evaluate the extent of interstitial fibrosis. Hamamatsu microscope was used to capture the whole tissue section and Aperio ImageScope software was used to quantify fibrosis in the left ventricular free wall remote from the infarction. Finally, sections were stained with FITC Labelled wheat germ agglutinin (WGA) to determine cardiomyocyte size. Cell size from transversally cut cardiomyocytes in the left ventricular free wall remote from the infarcted area was measured using image analysis (Zeiss KS400, Germany) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The investigators analysing the data were blinded to the treatment allocation.
Metabolic cage
Two weeks before termination, rats were placed in metabolic cages to monitor 24-hour water and food intake and 24-hour urine collection. The metabolic cages consist of an upper chamber with a grid floor where the rat is housed; and a lower chamber with specialized funnel that separates fecal pellets and urine. After 24-hour housed in the metabolic cage, under isoflurane anesthesia, blood samples were drawn from tail vein and plasma was collected. Plasma and urine were stored at -80 ˚C for later analysis.
Blood measurements
Blood samples were obtained via tail vein at regular intervals under isoflurane anaesthesia to monitor blood glucose and haematocrit levels. At sacrifice, 8 ml of blood was drawn from the abdominal aorta and urine was obtained from the bladder. Plasma creatinine and glucose were determined enzymatically on the Roche/Hitachi Cobas system (Roche Germany). Plasma sodium and potassium were measured by ion-selective electrode methods on the Roche/Hitachi Cobas system (Roche Germany). Plasma total ketone body concentrations were quantified via a cyclic enzymatic method using Autokit Total Ketone Body (Wako Chemicals, Germany).
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Urine measurementsUrine creatinine and glucose were determined enzymatically on the Roche/Hitachi cobas system (Roche Germany). Urine sodium and potassium were measured by ion-selective electrode methods on the Roche/Hitachi Cobas system (Roche, Germany). Urine total ketone body concentrations was quantified via a cyclic enzymatic method using Autokit Total Ketone Body (Wako Chemicals, Germany). Total urinary protein was measured by turbidimetric method on the Roche/Hitachi Cobas system (Roche Germany). Creatinine clearance, as an estimation for glomerular filtration rate, was calculated from urinary and plasma creatinine levels. Creatinine clearance = (urine creatinine x urine flow) / (plasma creatinine x body weight) and presented as mL/min/kg body weight.
Quantitative real-time polymerase chain reaction
RNA was extracted from the non-infarcted left ventricular free wall remote from the infarction using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) and the NanoDrop device was used to measure RNA concentration. Random primer mix was used to prepare first-stranded DNA and thereafter used as a template for quantitative real-time reverse-transcriptase-PCR (qRT-PCR) (25 ng/reaction). mRNA levels obtained by a qRT-PCR using C1000 Thermal Cycler CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories, Veenendaal, The Netherlands). 36B4 reference gene were used to correct all measured mRNA expression. Primer sequences can be found in supplementary Table S1.
